FDA is cell-membrane permeable and accumulates inside of viable cells as fluorescein (Fig. 1). Since fluorescein is less hydrophilic than BCECF or Calcein, the leakage of fluorescein from cells is rather high. FDA is also utilized for flow cytometry. The excitation and emission wavelengths of fluorescein are 488 nm and 530 nm, respectively. FDA-stained cells are shown in Fig. 2.
Fig. 1 Cell staining mechanism
1.Prepare 0.5 mg/ml FDA stock solution with DMSO. Dilute 10 ul of the stock solution with 5 ml PBS(-).
2.Prepare a cell suspension and wash cells with PBS(-). Prepare 1×105-1×106 cells/ml cell suspension
3.Add 15 ul FDA solution to 30 ul cell suspension, and incubate at 37ºC for 15-30 min.
4.Put 10 ul stained cell suspension on a glass slide and cover with a cover glass.
5.Observe the cells under a fluorescence microscope with 488 nm excitation and 530 nm emission filters.
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Fig. 2 Cell staining with FDA,
Cell type: HeLa